Plasmid transfection
SgRNA oligos (Supplementary Table 1) were annealed and cloned into
pX330-EGFP plasmid. HEK293 and B160F10 cells were transfected according
to the manufacturer’s protocols (Invitrogen, Cat. No. 11668-027). In
brief, HEK293 and murine B16-F10 cells were seeded on 12-well plates in
1ml of DMEM. When cells reached 60–80% confluency, medium was changed
to Opti-MEM. Cells were then transfected with Lipofectamine 2000
(Invitrogen) according to manufacturer’s protocol. One μg
pX330-sgRNAs and 2μg BE4 plasmids were mixed with 2μL
Lipofectamine 2000. Six hours later, medium was replaced with fresh
DMEM. Cells were then subjected to G418 treatment as described above.